Rapid infusion of autologous bone marrow derived stem cells

ABSTRACT

The present invention relates to a method and composition for the aspiration, processing, testing and infusion of bone marrow derived stem cells, as an adjuvant treatment in cardiovascular disorders. More specifically, the invention provides for the methods and compositions for the aspiration, analysis, processing, infusate preparation and infusion of bone-marrow derived stem cells, particularly in a rapid point-of-care environment, wherein a centrifugal fractionation and optically monitored separation of the bone marrow yield desired cellular product in the desired concentration and viscosity.

RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 14/296,360, filed Jun. 4, 2014; which is a continuationapplication of PCT Application No. PCT/US2014/010745, filed Jan. 8,2014; which claims priority to U.S. Provisional Application Ser. No.61/751,846, filed Jan. 12, 2013, all of which are hereby expresslyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a rapid point-of-care approach foraspirating, isolating, testing, and delivering a cell population andaccompanying factors obtained from bone marrow in a pre-determined doseand viscosity to a subject and compositions that have a cell population,which includes bone marrow stem cells and/or progenitor cells, ananticoagulant, and a viscosity of less than or equal to 5.0 centipoise(cP) measured at 37° C.

BACKGROUND OF THE INVENTION

Cardiovascular disease (CVD) is the number one cause of morbidity andmortality worldwide. An estimated 17.3 million people died from CVDs in2008, representing 30% of all global deaths. Of these deaths, anestimated 7.3 million were due to coronary heart disease and 6.2 millionwere due to stroke. More remarkably, low- and middle-income countriesare disproportionally affected, driving the need for regenerativetherapies in lieu of chronic drug treatment regimens and suchregenerative therapies must be offered in formats eliminating the needfor high cost laboratory infrastructure or extensive multi-hour usage ofvascular catheter labs. Over 80% of CVD deaths take place in low- andmiddle-income countries and occur almost equally in men and women. Inthe progression of CVDs, plaque lesions develop in arteries that resultin the narrowing of vessels, and in severe cases they break open andcreate a blockage of blood flow (ischemia) to a vital part of the heart,brain or limb. Such ischemia may be reversed if treated within a shortperiod of time by reperfusion therapy and further by the infusion ofadult tissue derived stem cells with or without the presence ofextracellular factors. Despite significant advances in medical therapyand revascularization strategies, the prognosis of certain patients withAcute Myocardial Infarction (AMI), Chronic Heart Failure (CHF), CriticalLimb Ischemia (CLI) and Ischemic Brain Injury (Stroke) remains dismalwithout the introduction of early biological repair intervention.

Along with reperfusion, adjuvant stem cell therapy has been shown to bepotentially efficacious in the repair and regeneration of damaged tissueof heart, brain and limbs from ischemic injury. These stem/progenitorcells can be isolated from different sources and one such source isbone-marrow. The autologous, bone-marrow derived in one case, peripheralblood derived in the second case, and adipose derived in the third,adult stem/progenitor cells circumvent the ethical and legal issuesrelated to embryonic stem cells. Further, it also terminates the risk oftransmitting diseases and immune rejection. The regenerative potentialof autologous stem cells, specifically adipose or peripheral blood, andmost specifically bone marrow derived cellular product is highlyinfluenced by the aspiration, processing, and delivery techniqueemployed. Anticoagulant plays a crucial role in the overall efficacy ofregenerative cell therapy, and extended exposure to anticoagulant hasbeen shown to have a negative impact on the “stemness” of the cells. Theuse of an anticoagulant in the aspiration syringe and processing deviceskeeps the bone-marrow in a non-coagulated state, which allows properstratification, isolation and infusion of stem/progenitor cells to treatclinical conditions. Anticoagulant also inhibits to varying degrees theformation of microthrombae, which if injected into the patient can causeadverse events. It is also understood that the addition of any chemicalentity in the presence of proteins or cells (“Biologicals”) may have aneffect on the structure or function of the Biologicals. Therefore, theanticoagulant used, the concentration used, and the time of cellexposure to the anticoagulant individually and potentially,cumulatively, plays a crucial role in the overall outcome of theefficacy and safety of stem cell therapy. The ideal stem cell compatibleanticoagulant must be in the optimal concentration ratio balance to keepthe cell therapy concentrate or infusate in the anti-coagulated statefor a defined period of time to complete the interventional procedureand also reduce or eliminate the intravascular formation of thrombaewhile minimizing the risk of peri-procedural bleeding and modulation ofstem cells.

Heparins are the most commonly used anticoagulant agents for bone-marrowaspiration. It has, however, been reported that heparin can interferewith the mobilization and homing mechanism(s) of stem and/or progenitorcells thereby impairing the functional therapeutic capacity of thesecells. Moreover, heparins are associated with high rates ofperi-procedural bleeding, which may be related to their inability tobind to clot-bound thrombin. Still further, heparins can get inactivatedin-vivo by binding to platelet factor-4 (PF-4). Certain individualsexpressing antibody to heparin-PF-4 complex can experienceHeparin-Induced-Thrombocytopenia (HIT) on exposure to heparins.

The anti-coagulated bone marrow aspirate is in a heterogenous mixturecontaining Hematopoietic Stem Cells (“HSCs”), Mesenchymal Stem Cells(MSCs), Endothelial Progenitor Cells (“EPCs), CXCR4 positive cells,White Blood Cells (“WBCs”), Red Blood Cells (“RBCs”), platelets(“PLTs”), plasma with serum proteins and metabolites (“PLASMA”), and fat(“FAT”) and is unsuitable for delivery in the raw aspirate form into apatient's vascular system. Furthermore, to properly define thetherapeutic cell mixture constituency for reproducible therapeuticefficacy patient-to-patient, and to prepare a safe but dose specificvolume of the therapeutic cell concentrate, the therapeuticallyeffective cells (HSC's, MSC's, WBC's, EPC's and CXCR4 positive cells)must be purified, isolated or captured from the ineffective andundesired cells and fat in a short period of time to render aconcentrated therapeutic dose of cells and factors at the point-of-care.

The purification of cells, and specifically WBCs containing the desiredHSC's, EPC's, MSC's and other stem/progenitors cells, has historicallybeen accomplished by placing the cells in a containment device andapplying standard centrifugal stratification under a gravity force usingeither chemical density based methods or in automated chemical freesystems. The cells of the bone marrow coming from the red marrow andstroma will stratify to their respective density under specific gravityforce (“g force”) in a defined period of time, and thus create layers ofcells in the containment device from bottom to top wherein the RBCs(most dense) are on the bottom, followed by the nucleated RBC's andVSELs, the WBC's, PLTs, PLASMA, and FAT. Both of the methods simplyharvest the buffy coat as typically defined as the cellular layer abovethe RBC/nucleated RBC fraction and below the PLT fraction.

The ideal cell purification process involves a closed minimallymanipulated procedure for rapidly stratifying and isolating the desiredHSCs, EPCs, MSCs plus other stem/progenitor cells, which may also bereferred to as stem cells or MNCs or progenitor cells or bone marrowconcentrate enriched in progenitor cells (BMC_(E)PC) throughout thisdocument, away from the undesired mature RBCs and topping up the desiredcell fraction with PLASMA to the proper volume and viscosity for safeand effective delivery into the vascular system via a stem cell friendlydevice such as an over the wire balloon catheter, or an intra-organdelivery cannula.

The delivery of stem cells via a catheter procedure is an ideal way ofinfusing cells proximally to the afflicted organ. However,trans-catheter passage of cells can potentially have a negative impacton the desired cells in several ways. First, the cells undergo varyingshear stresses depending on their proximity to the lumen wall, thevelocity of the injection, the radius of the lumen, and the viscosity ofthe fluid, and second since the lumen is a polymeric substrate itspotential to impact cells and cell membranes, which come in contactthrough activation, binding or shedding can be important.

The transcatheter delivery of stem cells into the vascular system isdesirably achieved by introduction of an intravascular catheter in theperipheral vasculature, and inducing transient ischemia in the targetedorgan proximally or distally to the catheter tip by inflating anintravascular balloon to slow or stop blood flow for a pre-determinedtime. Such ischemia improves the cellular homing of the stem andprogenitor cells to the ischemic tissue, and increases the opportunityfor the cells initiating one or more processes including but not limitedto tissue integration, cellular fusion, cellular differentiation, and/orparacrine factor release. Ischemia is best induced by inflating acompliant or non-compliant balloon to reduce or stop blood flow whiletaking extra care to minimize any endothelium wall damage from theballoon pressurization.

Thus, there is a need for an improved method and composition for theaspiration, processing, and delivery of bone marrow cells, such that thebone-marrow derived stem cells provide a safe and efficient adjuvanttherapy for re-vascularization and or organ repair in ischemicdisorders.

SUMMARY OF THE INVENTION

Fast and efficient methods and complete point-of-care kits foraspiration, processing, and infusion of autologous stem cells andfactors isolated from bone marrow in a period of less than 90 minuteshave been developed. In some embodiments, methods for bone-marrowharvesting and processing, comprise an amount or concentration of ananticoagulant, preferably bivalirudin, which minimizes clot formationfor up to 90 minutes without affecting the efficacy of stem celltherapy.

Some embodiments described herein yield a desired mixture comprisingstem and/or progenitor cells, which comprises buffy coat stem and/orprogenitor cells, such as hematopoietic stem cells (HSCs), mesenchymalstem cells (MSCs), endothelial progenitor cells (EPCs), and CXCR4positive cells with a reduced or lowered amount of RBC as compared toconventional cell populations obtained from bone marrow.

Some embodiments described herein provide for a rapid cellular infusateanalysis within the point-of-care environment, ensuring an adequate andviable dose for the cellular therapy.

Some embodiments described herein infuse, at a controlled viscosity andflow rate through either manual or metered mechanism(s), a cellpopulation having bone marrow-derived stem and/or progenitor cells at aviscosity, that is measured at normal human body temperature (e.g., aviscosity of 5.0 cP, 4.5 cP, 4.0 cP, 3.5 cP, 3.0 cP, 2.5 cP, 2.0 cP, 1.5cP, or 1.0 cP at 37° C.), so as to ensure that a viable and potent celldose can be delivered via a transcatheter injection while minimizingshear stress.

Some embodiments described herein infuse the aforementioned cellpopulation mixture through a transcatheter comprising control mechanismfor both proper and safe intravascular balloon inflation.

Some embodiments comprise an anticoagulant as the only added chemicalbeyond a sodium chloride buffer. In some embodiments the anticoagulantis bivalirudin. Unlike many anticoagulants, bivalirudin generates littleor no hemolysis.

Embodiments of the invention also include a disposable centrifugal cellstratification device, which may have controlled valving, capable ofresponding to motor driven on and off positioning or may have a densityfunctioning separation device such that bone marrow derived cellpopulations above or below the device may be harvested. Some embodimentscomprise an optional on-board firmware for controlling the interrogationand harvest of certain cell populations within a multi-stratified zonedriven by differences in a light source transmission value responding topre-determined light units or an equivalency density separation devicewhose density equals the targeted cells density to mass ratio, a rapidanalytical instrument verifying certain cell populations or cell doseand viscosity of certain populations, and an intravascularly placedballoon catheter where said balloon is inflated proximally to the targetorgan for a specified period stopping 80-100% blood flow to induceischemia. In some embodiments the controlled minimal cell shearinginfusion of harvested cells and factors through the cannula lumen at arate of 0.5 mL to 2.5 mL per minute as controlled or metered by a devicesuch as a mechanical pump or hand controlled method as interpreted by anin-line manual pressure meter results in a reduced degree of apoptosisor other cell death.

The disclosure is further summarized through reference to theembodiments listed below:

-   -   1. A composition comprising:        -   (a) a first cell population that comprises bone marrow stem            cells and/or progenitor cells,        -   (b) an anticoagulant, and        -   (c) an aqueous buffer and/or an autologous serum and/or an            autologous plasma fraction,    -   wherein the composition has a viscosity of not more than 5.0        centipoise (cP) measured at 37° C.    -   2. The composition of embodiment 1, wherein the first cell        population that comprises bone marrow stem cells and/or        progenitor cells is autologous.    -   3. The composition of embodiment 2, wherein the first cell        population that comprises bone marrow stem cells and/or        progenitor cells is from a medullary space within a bone of said        patient.    -   4. The composition of embodiment 3, wherein the bone is an iliac        crest, a femora, a tibiae, a spine, a sternum or a rib.    -   5. The composition of embodiment 3, wherein said first cell        population that comprises bone marrow stem cells are from the        medullary space within the iliac space.    -   6. The composition of any of embodiments 1-5, wherein said        anticoagulant is selected from the group consisting of a        coumarin, a vitamin K antagonist, an indirect thrombin        inhibitor, heparin, a factor Xa inhibitor, a direct thrombin        inhibitor, batroxobin, hemetin, a purified plant extract, EDTA,        citrate, oxalate, and a nitrophorin.    -   7. The composition of any of embodiments 1-6, wherein said        anticoagulant is a direct thrombin inhibitor.    -   8. The composition of any of embodiments 1-5, wherein said        anticoagulant comprises bivalirudin.    -   9. The composition of embodiment 8, wherein said bivalirudin is        provided from a stock solution having a concentration of 1 mg/mL        to 50 mg/mL.    -   10. The composition of embodiment 8, wherein said bivalirudin is        provided from a stock solution having a concentration of 5 mg/mL        to 35 mg/mL.    -   11. The composition of embodiment 8, wherein said bivalirudin is        provided from a stock solution having a concentration of 10        mg/mL to 25 mg/mL.    -   12. The composition of embodiment 8, wherein said bivalirudin is        provided from a stock solution having a concentration of 18        mg/mL to 22 mg/mL.    -   13. The composition of embodiment 8, wherein said bivalirudin is        provided from a stock solution having a concentration of 20        mg/mL.    -   14. The composition of embodiment 8, wherein said bivalirudin is        present in said composition at a concentration of 1 mg/mL-3        mg/mL.    -   15. The composition of embodiment 8, wherein said bivalirudin is        present in said composition at a concentration of 1.5 mg/mL-2.5        mg/mL.    -   16. The composition of embodiment 8, wherein said bivalirudin is        present in said composition at a concentration of 1.8 mg/mL-2.2        mg/mL.    -   17. The composition of embodiment 8, wherein said bivalirudin is        present in said composition at a concentration of 2.0 mg/mL.    -   18. The composition of any of embodiments 1-17, wherein said        viscosity measured at 37° C. is 1.0 cP-5.0 cP.    -   19. The composition of any of embodiments 1-17, wherein said        viscosity measured at 37° C. is 1.8 cP-3.0 cP.    -   20. The composition of any of embodiments 1-17, wherein said        viscosity measured at 37° C. is 2.0 cP-2.5 cP.    -   21. The composition of any of embodiments 1-17, wherein said        viscosity measured at 37° C. is 3.1 cP-5.0 cP.    -   22. The composition of any of embodiments 1-21, wherein said        aqueous buffer comprises an ionic salt.    -   23. The composition of any of embodiments 1-21, wherein said        aqueous buffer comprises sodium chloride.    -   24. The composition of any of embodiments 1-21, wherein said        aqueous buffer comprises sodium chloride and the amount of said        sodium chloride in said composition is 0.9%.    -   25. The composition of any of embodiments 1-24, wherein said        cell population that comprises bone marrow cells comprises        mononuclear cells that comprise at least a stem cell and/or        progenitor cell selected from the group consisting of        hematopoietic stem cells, mesenchymal stem cells, endothelial        progenitor cells, and CXCR4 positive cells.    -   26. The composition of any of embodiments 1-25, wherein said        cell population that comprises bone marrow cells comprises at        least 10⁷ mononuclear cells.    -   27. The composition of any of embodiments 1-25, wherein said        cell population that comprises bone marrow cells comprises not        more than 10⁹ mononuclear cells.    -   28. The composition of any of embodiments 1-25, wherein said        cell population that comprises bone marrow cells comprises at        least 10⁴ hematopoietic stem cells.    -   29. The composition of any of embodiments 1-25, wherein said        cell population that comprises bone marrow cells comprises at        least 5×10² mesenchymal stem cells.    -   30. The composition of any of embodiments 1-25, wherein said        cell population that comprises bone marrow cells comprises at        least 5×10² endothelial progenitor cells.    -   31. The composition of any of embodiments 1-25, wherein said        cell population that comprises bone marrow cells comprises at        least 5×10³ CXCR4 positive cells.    -   32. The composition of any of embodiments 1-31, wherein said        cell population that comprises bone marrow cells comprises at        least 10⁷ to not more than 10⁹ mononuclear cells, at least 10⁴        hematopoietic stem cells, at least 5×10² mesenchymal stem cells,        at least 5×10² endothelial progenitor cells, and at least 5×10³        CXCR4 positive cells.    -   33. The composition of any of embodiments 1-32, wherein said        composition demonstrates an apoptosis or other cell death rate        of not more than about 40% one hour after delivery at a flow        rate of 2.5 mL/min, through a catheter having a lumen size of        approximately 0.36 mm and using a 5 mL syringe holding pressure        to 28.39 psi with a plunger force of 4.85 lbf.    -   34. The composition of any of embodiments 1-32, wherein said        composition demonstrates an apoptosis or other cell death rate        of not more than about 40% one hour after delivery through a        catheter if subjected to a maximum shear of not more than        9101/second.    -   35. A method of making a regenerative cell composition        comprising:        -   (a) providing a first cell population that comprises bone            marrow stem cells and/or progenitor cells;        -   (b) mixing an anticoagulant with said first cell population            so as to produce a composition comprising said first cell            population and anticoagulant;        -   (c) enriching bone marrow stem cells within said composition            comprising said cell population and anticoagulant so as to            produce a second cell population that comprises enriched            bone marrow stem cells and/or progenitor cells;        -   (d) isolating a fraction comprising said second cell            population that comprises enriched bone marrow stem cells            and/or progenitor cells; and        -   (e) adjusting the viscosity of said fraction using a liquid            to a viscosity of not more than 5.0 cP measured at 37° C. so            as to make said regenerative cell composition.    -   36. The method of embodiment 35, wherein said first cell        population that comprises bone marrow stem cells and/or        progenitor cells comprises aspirated cells from bone marrow.    -   37. The method of embodiment 36, wherein said aspirated cells        from bone marrow are aspirated through an aspiration needle        having a needle/trocar of 8 to 14 gauge.    -   38. The method of embodiment 36, wherein said aspirated cells        from bone marrow are aspirated through an aspiration needle        having a needle/trocar of 10 to 12 gauge.    -   39. The method of embodiment 36, wherein said aspirated cells        from bone marrow are aspirated through an aspiration needle        having a needle/trocar of 11 gauge.    -   40. The method of any of embodiments 35-39, wherein said        aspirated cells are aspirated through a needle/trocar at        negative pressure of less than 20 psi.    -   41. The method of any of embodiments 35-39, wherein said        aspirated cells are aspirated through a needle/trocar at        negative pressure of less than 15 psi.    -   42. The method of any of embodiments 35-39, wherein said        aspirated cells are aspirated through a needle/trocar at        negative pressure of less than 10 psi.    -   43. The method of any of embodiments 35-39, wherein said        aspirated cells are aspirated through a needle/trocar at        negative pressure of less than 5 psi.    -   44. The method of embodiment 35, wherein said liquid is an        autologous plasma fraction.    -   45. The method of embodiment 35, wherein said liquid is an        autologous serum or plasma fraction comprising liquid from which        said fraction comprising said second cell population that        comprises enriched bone marrow stem cells and/or progenitor        cells was isolated.    -   46. The method of embodiment 35, wherein said anticoagulant is        contacted with said first cell population that comprises bone        marrow stem cells and/or progenitor cells prior to obtaining        said first cell population that comprises bone marrow stem cells        and/or progenitor cells from a subject.    -   47. The method of embodiment 35, wherein said anticoagulant is        contacted with said first cell population that comprises bone        marrow stem cells subsequent to obtaining said first cell        population that comprises bone marrow stem cells from said        subject.    -   48. The method of embodiment 47, wherein the anticoagulant is        mixed with said first cell population at least in part by        manually agitating a container containing the first cell        population and the anticoagulant.    -   49. The method of embodiment 48, wherein the agitating comprises        rolling the container.    -   50. The method of any of embodiments 35-49, wherein the        anticoagulant comprises bivalirudin.    -   51. The method of embodiment 50, wherein said bivalirudin is        provided at a concentration of 1 mg/mL-3 mg/mL.    -   52. The method of embodiment 50, wherein said bivalirudin is        provided at a concentration of 1.8 mg/mL-2.2 mg/mL.    -   53. The method of embodiment 50, wherein said bivalirudin is        provided at a concentration of 2.0 mg/mL.    -   54. The method of any of embodiments 35-49, wherein said        viscosity measured at 37° C. is 1.0 cP-5.0 cP.    -   55. The method of any of embodiments 35-49, wherein said        viscosity measured at 37° C. is 1.8 cP-3.0 cP.    -   56. The method of any of embodiments 35-49, wherein said        viscosity measured at 37° C. is 3.1 cP-5.0 cP.    -   57. The method of any of embodiments 35-49, wherein said liquid        is an aqueous buffer that comprises an ionic salt.    -   58. The method of embodiment 57, wherein said aqueous buffer        comprises sodium chloride.    -   59. The method of embodiment 58, wherein the amount of said        sodium chloride in said regenerative cell composition is 0.9%.    -   60. A method of ameliorating ischemia or a condition associated        with ischemia in a subject comprising:    -   identifying a subject having an ischemia or a condition        associated with ischemia;    -   providing to said subject a composition comprising:        -   (a) a first cell population that comprises bone marrow stem            cells and/or stem cell progenitor cells,        -   (b) an anticoagulant, and        -   (c) an aqueous buffer and/or an autologous serum and/or an            autologous plasma fraction,    -   wherein the composition has a viscosity of not more than 5.0        centipoise (cP) measured at 37° C.; and    -   determining an improvement in ischemia or said condition        associated with ischemia, such as by measuring or observing in        said subject an increased blood flow, an improvement in left        ventricle ejection fraction, an improvement in cardiac output, a        reduction in cardiac scar formation, an improvement in cardiac        remodeling, an increase in angiogenesis, an increase in        vascularity, an improvement in neural scar formation, or an        increase in amputation-free survival rate.    -   61. The method of embodiment 60, wherein the composition is        provided to said subject not more than 2 hours after said first        cell population that comprises stem cells and/or progenitor        cells is obtained.    -   62. The method of embodiment 60, wherein the composition is        provided to said subject not more than 90 minutes after said        first cell population that comprises bone marrow stem cells        and/or progenitor cells is obtained.    -   63. The method of embodiment 60, wherein the composition is        provided to said subject not more than 60 minutes after said        first cell population that comprises bone marrow stem cells        and/or progenitor cells is obtained.    -   64. The method of any of embodiments 60-63, wherein the blood        flow at an ischemic tissue of said subject is blocked for 1-3        minutes.    -   65. The method of any of embodiments 60-64, wherein the blood        flow at an ischemic tissue of said subject is blocked for 2        minutes.    -   66. The method of any of embodiments 64-65, wherein said blood        flow is blocked by an inflatable balloon.    -   67. The method of embodiment 66, wherein said balloon is        inflated to block at least 80% of blood flow distal to said        balloon.    -   68. The method of any of embodiments 60-67, wherein said        composition is provided to said subject at a rate of no more        than 2.5 mL per minute.    -   69. The method of any of embodiments 60-68, wherein said        composition provided to said subject is not subjected to a        maximum shear of more than 9101/second.    -   70. The method of any of embodiments 60-69, wherein said        composition is provided to said subject through a catheter        having a lumen size of approximately 0.36 mm.    -   71. The method of any of embodiments 60-70, wherein said        composition is provided to said subject at a dose of 5 mL per        dose.    -   72. The method of any of embodiments 60-70, wherein said        composition is provided under a set of parameter conditions not        exceeding those of Parameter Set 1 of Table 2.    -   73. The method of any of embodiments 60-70, wherein said        composition is provided under a set of parameter conditions not        exceeding those of Parameter Set 2 of Table 2.    -   74. The method of any of embodiments 60-70, wherein said        composition is provided under a set of parameter conditions not        exceeding those of Parameter Set 3 of Table 2.    -   75. The method of any of embodiments 60-70, wherein said        composition is provided under a set of parameter conditions not        exceeding those of Parameter Set 4 of Table 2.    -   76. The method of any of embodiments 60-70, wherein said        composition is provided under a set of parameter conditions not        exceeding those of Parameter Set 5 of Table 2.    -   77. The method of any of embodiments 60-70, wherein said        composition is provided under a set of parameter conditions not        exceeding those of Parameter Set 6 of Table 2.    -   78. The method of any of embodiments 60-71, wherein said        composition is provided as a single dose.    -   79. The method of any of embodiments 60-71, wherein said        composition is provided as at least two doses.    -   80. The method of any of embodiments 60-71, wherein said        composition is provided as four doses of 5 mL each.    -   81. The method of any of embodiments 60-71, wherein said        composition is provided as a single dose per day on each of        multiple days.    -   82. The method of any of embodiments 60-71, wherein said        composition is provided as at least two dose per day on each of        multiple days.    -   83. The method of any of embodiments 60-82, wherein providing to        said subject a composition is followed by a resting period of        not more than 5 minutes following each dose provided.    -   84. The method of any of embodiments 60-82, wherein providing to        said subject a composition is followed by a resting period of        not more than 4 minutes following each dose provided.    -   85. The method of any of embodiments 60-82, wherein providing to        said subject a composition is followed by a resting period of        not more than 3 minutes following each dose provided.    -   86. The method of any of embodiments 60-82, wherein providing to        said subject a composition is followed by a resting period of        not more than 2 minutes following each dose provided.    -   87. The method of any of embodiments 60-82, wherein providing to        said subject a composition is followed by a resting period of        not more than 1 minute following each dose provided.    -   88. The method of any of embodiments 83-87, wherein a last        resting period is followed by a 3 mL dose of a chaser solution        to evacuate any remaining cells at a rate not exceeding 2.5 mL        per minute.    -   89. The method of any of embodiments 60-86, wherein said        composition is provided intravascularly.    -   90. The method of any of embodiments 60-86, wherein said        composition is provided intramuscularly.    -   91. The method of any of embodiments 60-86, wherein said        composition is provided intra-arterially.    -   92. The method of any of embodiments 60-86, wherein said        composition is provided intra-coronarily.    -   93. The method of any of embodiments 60-92, wherein said        composition is provided using a catheter coated with a        biocompatible material.    -   94. The method of any of embodiments 60-93, wherein said        composition comprises any of the compositions of embodiments        1-34.    -   95. The method of any of embodiments 60-93, wherein said        composition comprises any of the regenerative cell compositions        made by the methods of embodiments 38-61.    -   96. The method of any of embodiments 60-95, wherein the ischemia        is cardiac ischemia.    -   97. The method of any of embodiments 60-95, wherein the ischemia        is critical limb ischemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the treatment approach, achieving adesirable time frame for any bedside therapeutic in the heartcatheterization procedure.

FIG. 2A: One section of the cardiac MR stacking used to calculatemyocardial volume at 3 months post-BMC_(E)PC infusion.

FIG. 2B: Left Ventricle transmurality index at 3 months post-BMC_(E)PCinfusion.

FIG. 2C: One section of the cardiac MR stacking used to calculatemyocardial volume at 24 months post-BMC_(E)PC infusion.

FIG. 2D: Left Ventricle transmurality index at 24 months post-BMC_(E)PCinfusion.

FIG. 3A: CT Angiogram (representative example) of a pre-treated (leftleg) of subject number EHIRC/CLI-STEM-002 on enrollment in studydemonstrating poor vascularity.

FIG. 3B: CT Angiogram (representative example) of 12 monthspost-treatment (left leg) of subject number EHIRC/CLI-STEM-002,demonstrating improvement of vascularity over FIG. 3A, above.

DETAILED DESCRIPTION

Aspects of the embodiments disclosed herein include methods of makingcell populations that are significantly improved over conventional cellpopulations for amelioration or improvement of ischemic conditions(e.g., cardiac ischemia, limb ischemia). Particular formulations of cellpopulations and methods of use thereof have been generated, whichunexpectedly improve ischemic conditions (e.g., greater than 4-foldimprovement in left ventricle ejection fraction (LVEF) after cardiacischemia, as compared to conventional approaches, and greater than 25%improvement in amputation-free survival rate after critical limbischemia). It has been discovered that conventional cell populationshaving stem and progenitor cells do not have an appropriate viscosity,and/or anticoagulant, and are not delivered to the subject in a mannerthat improves or promotes cell viability. Accordingly, aspects of theinvention relate to compositions comprising a first cell population thatcomprises bone marrow stem and progenitor cells, and anticoagulant, andan aqueous buffer and/or an autologous serum and/or an autologous plasmafraction, wherein the composition has a viscosity of not more than 5.0centipose (cP) measured at 37° C., and methods of delivery such that thecomposition is provided to a subject at a rate of not more than 2.5mL/minute.

Aspects of the embodiments disclosed herein include methods for bonemarrow aspiration, which comprise a) preparation of bivalirudinsolution, b) filling of syringes with anticoagulant solution, and c)aspiration of bone marrow into the syringe containing the saidanticoagulant solution. The said method for bone-marrow harvesting andprocessing, further comprises an amount of an anticoagulant, whichminimizes clot formation up to 90 minutes without affecting the efficacyof stem cell therapy and minimizing adverse pen-procedure bleeding orclotting events. Another important aspect of some embodiments is theavoidance of using any xenobiotic or citrate agents in the processing ofbone marrow.

Aspects of the embodiments disclosed herein include compositionscomprising bone marrow stem cells and/or progenitor cells. In somecontexts, with respect to particular embodiments, the phrase ‘bonemarrow stem cells and/or progenitor cells’ is meant to encompass bonemarrow cell populations that may differentiate into a specific celltype, independent of whether they may replicate indefinitely or not,and/or produce growth factors, and/or facilitate, contribute, or promoteblood vessel formation, an increase in blood vessel diameter, and/or anincrease in blood flow in a tissue (e.g., an ischemic tissue).

Yet more embodiments concern the rapid analysis and an improvedtechnique for bone marrow processing thereby yielding a defined mixtureof desired cellular product, and if required the addition of plasma as adiluent, within limits of the intended dosage and viscosity. The initialcell count of harvested bone marrow and processed cellular product wouldbe rapidly performed at the patient's bedside. The said mixture ofcellular product comprises, but not limited to, Hematopoietic stem cells(HSC) and/or Hematopoietic progenitor cells (HPC) and/or CD34 positivecells, identified following ISHAGE guidelines for CD34 positive cellenumeration; Mesenchymal stem cells (MSC) and/or stromal cells,identified as at least lineage negative/dim, CD45 negative/dim and CD73positive cells; Endothelial progenitor cells (EPC), identified as atleast CD45 negative/dim, CD34 positive and vascular endothelial growthfactor receptor 2 (VEGFR2) positive cells; CXCR4 progenitor cellsidentified as at least lineage negative/dim, CD45 negative/dim, andCD184 (CXCR4) positive cells. The said mixture of progenitor cellsshould also possess colony forming capacity as assessed by colonyforming unit assays for HSC/HPCs, MSCs/stromal cells and EPCs. Theaforementioned cell population comprises a variety of cell types thatwork synergistically in the overall healing of the injured tissue.

Along with the composition, the dosage and dosing method of the stemcells infusion are embodiments. A rapid testing of the cellular mixtureis performed in a point-of-care environment, ensuring a range of 10⁷ to10⁹ Mononuclear cells (MNC), including HSC not less than 10⁴ per dose,MSC not less than 5×10² per dose, EPC not less than 5×10² per dose,CXCR4 positive cells not less than 5×10³ per dose and a final viscositymeasured at 60° Fahrenheit to 98.6° Fahrenheit and delivered at normalbody temperature between 1.0 to 5.0 centipoise (cP) as titrated withautologous PLASMA in the cellular product. The mode of administration ofthe processed cellular mixture is further infused intramuscularly and/orintravascularly at a rate of 0.5 mL to 2.5 mL per minute, as an adjuvanttherapy. The said intravascular route further comprises intra-arterialsuch as intra-coronary by employing a catheter having at least twoindependent lumens one of which delivers the cells and is coated with abiocompatible or bio-inert polymer having an inner diameter between0.012″ to 0.019″ and the other lumen for inflating a polymeric balloonfor restricting blood flow; intra-venous; and the intramuscular mode ofadministration comprises intra-myocardial, intra-epicardial,intra-muscular and so on.

The source material for the compositions and methods disclosed hereinmay be bone marrow. Other stem cell sources are contemplated. The bonemarrow from which stem cells are isolated can be selected from the groupcomprising iliac crest, femora, tibiae, sternum, spine, ribs or othermedullary spaces in bone. A preferred bone marrow source is the iliaccrest. In some embodiments the stem and/or progenitor cell source suchas the bone marrow stem cell source is autologous to the patient whowill ultimately receive the composition. In some embodiments, beforestarting the aspiration of bone marrow, the anticoagulant solution isfilled into aspiration syringes. A number of anticoagulant sources arecontemplated, for example a coumarin, a vitamin K antagonist, anindirect thrombin inhibitor, heparin, a factor Xa inhibitor, a directthrombin inhibitor, batroxobin, hemetin, a purified plant extract, EDTA,citrate, oxalate, and a nitrophorin. A preferred anticoagulant isbivalirudin.

Bivalirudin has a number of beneficial properties, such as a short (20minute) half-life and a mode of action which is independent of manyother anticoagulants, such as heparin, which may be independentlyadministered to the patient pursuant to surgical intervention throughwhich stem cell sources such as bone marrow sources are obtained. Insome embodiments the concentration of anticoagulant, such asbivalirudin, to be used is 0.9 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL,12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, 25 mg/mL, 26mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 31 mg/mL, 32 mg/mL, 33mg/mL, 34 mg/mL, 35 mg/mL, 36 mg/mL, 37 mg/mL, 38 mg/mL, 39 mg/mL, 40mg/mL, 41 mg/mL, 42 mg/mL, 43 mg/mL, 44 mg/mL, 45 mg/mL, 46 mg/mL, 47mg/mL, 48 mg/mL, 49 mg/mL, 50 mg/mL, 51 mg/mL, 52 mg/mL, 53 mg/mL, 54mg/mL, or 55 mg/mL. In some embodiments the concentration ofanticoagulant, such as bivalirudin, to be used falls in the range of 1mg/mL to 50 mg/mL, preferably 5 mg/mL to 35 mg/mL, more preferably 10mg/mL to 25 mg/mL, most preferably 20 mg/mL. Illustratively, each vialmay contain 250 mg of bivalirudin, to which 5 mL of sterile water isadded for injection. One may gently swirl the anticoagulant such asbivalirudin with the water, for example until all material is dissolvedand the solution appears clear. One may then aspirate the solution intoa sterile syringe such as a 20 mL sterile syringe. One may then fill thesyringe with 0.9% Sterile sodium chloride, for example, for injection upuntil 12.5 mL mark, to yield a final concentration of 20 mg/mL. One maythen take three sterile 20 mL syringes and fill 2 mL of 20 mg/mLbivalirudin solution. One may then aspirate 18 mL of stem cell source ineach syringe to make a final volume of 20 mL. The final concentration ofthe anticoagulant, such as bivalirudin, along with aspirated bonemarrow, in each syringe may be 2 mg/mL. It is known to those skilled inthe art of aspiration of bone marrow that the aspiration needle used is7, 8, 9, 10, 11, 12, 13, 14, or 15 gauges, such as in the range of 8 to14 gauges, such as such as in the range of 9 to 12 gauges, preferablythe needle/trocar is of 11 gauges. The stem and/or progenitor cellsource, such as bone marrow is aspirated in the syringe or syringespre-filled with anticoagulant solution, for example for a finalcumulative volume between 120 mL to 180 mL. The aspiration syringe usedmay have a volume of 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL,12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41 mL, 42mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, 50 mL, 51 mL, 52mL, 53 mL, 54 mL, 55 mL, 56 mL, 57 mL, 58 mL, 59 mL, 60 mL, 61 mL, 62mL, 63 mL, 64 mL, 65 mL, 66 mL, 67 mL, 68 mL, 69 mL, 70 mL, 71 mL, 72mL, 73 mL, 74 mL, 75 mL, 76 mL, 77 mL, 78 mL, 79 mL, 80 mL, 81 mL, 82mL, 83 mL, 84 mL, 85 mL, 86 mL, 87 mL, 88 mL, 89 mL, 90 mL, 91 mL, 92mL, 93 mL, 94 mL, 95 mL, 96 mL, 97 mL, 98 mL, 99 mL, 100 mL, 101 mL, 102mL, 103 mL, 104 mL, 105 mL, 106 mL, 107 mL, 108 mL, 109 mL, or 110 mL,preferably may fall in the range of 5 mL to 100 mL, preferably 10 to 60mL, more preferably 20 to 50 mL, most preferably 20 mL.

Modifications to the above method may be made without departing from theinvention. For example, entry may be made into more than one-site toaspirate bone marrow. Some amount of anti-coagulant solution may beinjected into bone marrow before aspirating it. In the next step, theaspirated bone marrow is thoroughly mixed with anticoagulant solution byrolling gently between the palms of hands. The bone marrow is thentransferred into a device for separating the components of bone marrow,the art of which is known.

The stem and/or progenitor cell source, such as bone marrow, may beremoved from the patient or subject, for example by aspiration orexcision. In a preferred embodiment, the stem cell source, such as bonemarrow, is removed via aspiration. The stem cell source, such as bonemarrow, for example aspirated bone marrow, may be autologous, and may beanalyzed (both at pre- and post-processing step) and processed, in apoint-of-care environment. As an example of removal and examination at apoint-of-care environment, initially 120 mL of anti-coagulatedbone-marrow is harvested. Nucleated cells present per ml of sample arerapidly analyzed using a small sample (less than 0.5 mL) of it. Ifneeded 30-60 mL of additional bone marrow sample is harvested forprocessing in order to get the desired infusion cell dose. In someembodiments the processing technology comprises the followingcomponents; (i) a single sterile, disposable bone marrow processingdevice that can process variable volumes of bone-marrow up to 200 mL,(ii) a control module having at least one gravitational and one or moreInfra-red (IR)/optical sensors coupled with a microprocessor and motorsthat control the flow, movement, and compartmentalization of theseparated fractions into a disposable device or compartment, (iii) adocking station for the control module that transfers processing datafrom the control module firmware to a computer and (iv) a centrifugewith programmable parameters. In some embodiments, the purificationdevice comprises at least one of the components recited above. Throughpractice of the methods herein, one may isolate stem and progenitorcells from bone marrow, so as to produce at least one of a stem cellfraction, a plasma fraction, and a red blood cell fraction, preferablyin less than 30 minutes at the patient's bedside, which may also serveas the source of the autologous plasma. In some embodiments, a stem cellfraction, plasma fraction, and a red blood cell fraction may differ intheir viscosities. In some embodiments, a stem cell fraction may have aviscosity of above 5.0 cP at 37° C., and a plasma may have a viscosityof below 5.0 cP at 37° C., such as substantially below 5.0 cP at 37° C.,such that one may in some embodiments add a volume of the plasma to thestem cell fraction having a viscosity of above 5.0 cP at 37° C., suchthat the composition comprising the stem cell fraction a volume of theplasma may have a viscosity of 5.0 cP or lower at 37° C. As beneficiallydisclosed herein, the concentrated stem cells infusate (BMC_(E)PC)viscosity is titrated to ensure shear stresses are controlled duringtrans-catheter delivery. A viscometer, possibly including but notrequired ancillary software, may be used to determine the stem cellconcentrate viscosity in units (for example, centipoise(cP)), and tocalculate the required plasma volume required to yield a final infusatein a preferred range of 1.8-5 cP at 98.6° Fahrenheit or 37° Celsius. Apreferred infusate viscosity is 3.1 cP-5.0 cP, and a most preferredinfusate viscosity is 1.8 cP-3.0 cP, and a most preferred infusateviscosity is 2.0 cP to 2.5 cP, and anything beyond 5.0 cP is notpreferred. In some embodiments, the infusate viscosity is less than orequal to or any number in between 1.8 cP, 1.9 cP, 2.0 cP, 2.1 cP, 2.2cP, 2.3 cP, 2.4 cP, 2.5 cP, 2.6 cP, 2.7 2.8 cP, 2.9 cP, 3.0 cP, 3.1 cP,3.2 cP, 3.3 cP, 3.4 cP, 3.5 cP, 3.6 cP, 3.7 cP, 3.8 cP, 3.9 cP, 4.0 cP,4.1 cP, 4.2 cP, 4.3 cP, 4.4 cP, 4.5 cP, 4.6 cP, 4.7 cP, 4.8 cP, 4.9 cP,or 5.0 cP.

The stem cell source, such as bone marrow, may be stratified using adevice such as a disposable centrifugal cell stratification device,which may have controlled valving capable of responding to motor drivenon and off positioning and may have a density functioning separationdevice such that bone marrow derived cell populations above or below thedevice may be harvested, although variations on each parameter of adevice consistent with the parameters mentioned above, and alternativescomprising the use of alternative devices or for which cells arestratified without the use of a device as disclosed herein are alsocontemplated. Some embodiments comprise an optional on-board firmwarefor controlling the interrogation and harvest of certain cellpopulations within a multi-stratified zone driven by differences in alight source transmission value responding to pre-determined light unitsor an equivalency density separation device whose density equals thetargeted cells density to mass ratio, a rapid analytical instrumentverifying certain cell populations or cell dose and viscosity of certainpopulations, and an intravascularly placed balloon catheter where saidballoon is inflated proximally to the target organ for a specifiedperiod stopping 80-100% blood flow to induce ischemia. In someembodiments, the controlled minimal cell shearing infusion of harvestedcells and factors through the cannula lumen at a rate of 0.5 mL to 2.5mL per minute as controlled or metered by a device, such as a mechanicalpump or hand controlled method, as interpreted by an in-line manualpressure meter results in a reduced degree of apoptosis or other celldeath. In some embodiments, cell fractionation is beneficially attainedupon fractionation at 2000×g high speed spin for 15 minutes, followed by80×g low speed spin for five minutes, or variants thereupon, such as afirst spin of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 minutes, a first g-force of 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, or 2500, a second spin for 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 minutes at a g-force of 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, or 160.

The composition generated hereby, such as a cell population comprisingbone marrow stem cells, for example an aspirated and processed bonemarrow-derived stem cell population, may be infused as an adjuvant toprimary care, such as primary care in response to ischemia (e.g.,cardiac ischemia and/or limb ischemia). The stem cell compositionconcentrate, for example an aspirated and processed bone marrow-derivedstem and progenitor cell population, with or without factors, isinfused, in some embodiments, shortly after the tissue becomes ischemic.In some embodiments the stem cell composition concentrate, for examplean aspirated and processed bone marrow-derived stem cell population,with or without factors, is infused after ischemic conditioning of thetissue. In some embodiments, ischemic conditioning is effected byobstructing such as stopping blood flow to the affected area, forexample using an over the wire inflatable balloon inflated to reduceblood flow, or other reversibly active obstructant of blood flow toobstruct or impede blood flow distally to the balloon. In preferredembodiments the obstructant such as a wire guided inflated balloonobstructs 80-99.99% of blood flow, such as greater than or equal to anynumber in between 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%, orfor a period of no more than three minutes, preferably one minute andmost preferred for two minutes. Following a short period of tissueischemia such as period of no more than three minutes, preferably oneminute and most preferred for two minutes, cells are infused at acontrolled rate, no more than 2.5 mL per minute, such as 0.5 mL perminute, 0.6 mL per minute, 0.7 mL per minute, 0.8 mL per minute, 0.9 mLper minute, 1.0 mL per minute, 1.1 mL per minute, 1.2 mL per minute, 1.3mL per minute, 1.4 mL per minute, 1.5 mL per minute, 1.6 mL per minute,1.7 mL per minute, 1.8 mL per minute, 1.9 mL per minute, 2.0 mL perminute, 2.1 mL per minute, 2.2 mL per minute, 2.3 mL per minute, 2.4 mLper minute, or up to 2.5 mL per minute.

It is beneficially disclosed that infusion at a high shear rate mayinduce apoptosis/cell death in a substantial fraction of the compositiongenerated hereby, such as a stem cell composition, for example anaspirated and processed bone marrow-derived stem and/or progenitor cellpopulation. Accordingly, in some embodiments a shear rate of 9101/secondis an upper limit for the shear to which cells are exposed. Shear rateis a function of catheter diameter, and flow rate. Beneficiallydisclosed herein is the observation that a catheter with a approximately0.36 mm, or 0.014″ diameter and length of 1550 mm at a maximum shearrate of 9101/second should not exceed infusion above 2.5 mL/min with abone marrow viscosity of 5 cP. Alternately, at a viscosity ofapproximately 4 cps at the preferred flow rate of 2.5 mL/min one mayobserve a loss, for example through apoptosis or other cell death, of40% of the MNCs one hour post-infusion. Accordingly, a beneficial set ofparameters for the infusion of a composition generated hereby, such as astem cell composition, for example an aspirated and processed bonemarrow-derived stem and progenitor cell population, used is infused asan adjuvant to primary care, such as primary care in response toischemia, may comprise a composition viscosity from 1.6 to 5 cP, flowrate of 0.5 mL to 2.5 mL/min, lumen size approximately 0.36 mm and usinga 5 mL syringe we could hold the psi to 3.4 to 44.64 with a plungerpressure of 0.58 lbf to 25.0 lbf and minimize the apoptosis or othercell death in said composition. It is observed using manual delivery ofinfusate with higher-viscosity compositions may be more sensitive to theapplication of variable plunger forces. Accordingly, in some embodimentsuse of a 5 mL syringe is preferred as in some embodiments it may lowerthe force required for a given pressure compared to syringes of largervolume and in some embodiments may be beneficial, for exampleergonomically beneficial, for promoting more consistent control of thedelivery rate.

A study of the impact of flow rate, viscosity, pressure, syringe sizeand plunger force on shear rate is given in Table 1, below.

TABLE 1 Fluid Plunger Plunger Plunger Lumen Inner Wall Shear Wall Shearviscosity force (lbf) force (lbf) force (lbf) diameter Flaw rate Flowrate rate (WSR)/ Stress (centapoise, Pressure 5 mL 10 mL 20 mL S. no.(mm) (mL/min) (uL/sec) second (dyne/cm2) cP) (psi) syringe syringesyringe 1 0.36 1 16.667 3640 72.81 2 8.928 1.526688 2.258784 3.919392 20.36 1.25 20.83375 4550 91.013 2 11.16 1.90836 2.82348 4.89924 3 0.361.5 25.0005 5460 109.215 2 13.392 2.290032 3.388176 5.879088 4 0.36 1.7529.16725 6370 127.418 2 15.624 2.671704 3.952872 6.858936 5 0.36 233.334 7281 145.62 2 17.856 3.053376 4.517568 7.838784 6 0.36 2.2537.50075 8191 163.823 2 20.088 3.435048 5.082264 8.818632 7 0.36 2.541.6675 9101 182.025 2 22.32 3.81672 5.64696 9.79848 8 0.36 2.7545.83425 10011 200.228 2 24.552 4.198392 6.211656 10.77833 9 0.36 350.001 10921 218.43 2 26.784 4.580064 6.776352 11.75818 10 0.36 3.2554.16775 11831 236.633 2 29.016 4.961736 7.341048 12.73802

For a given maximum shear of 9101/second, a range of viscosities,pressures, syringe sizes and plunger forces are given in Table 2, below.

TABLE 2 Parameter Viscosity Syringe Plunger Set (cP) Pressure (Psi) size(mL) force (lbf) 1 2 22.32 5 3.81672 2 4 44.64 5 7.63344 3 2 22.32 105.64696 4 4 44.64 10 11.29392 5 2 22.32 20 9.79848 6 4 44.64 20 19.59696

Impact of different flow rates and viscosities on the cell health andpotency are given in Table 3, below.

TABLE 3 Dead and Apoptotic % Change in MNC cells (%)-Annexin-V % CellViability Average Pressure counts 1 h post- % Change in Colony Sampletype % Live Cells Assay (7AAD/PI) (Psi) Viscosity (cP) incubationforming units (CFUs) Pre-processed 90.30 9.70 94.02 — 1.6-1.8 — —(untreated cells) VXP processed 83.50 16.50 94.4 — 1.6-1.8 Baseline —Post-Catheter (1 mL/min) 79.60 20.40 95.35 6.8 1.6-1.8 No change —Post-Catheter 75.2 24.8 95.02 20 1.6-1.8 −10.08 — (2.5 mL/min) 26.13.1-5.0 — −17.43 Post-Catheter 75.70 24.30 91.3 42 1.6-1.8 −33.92 — (5mL/min)

In some embodiments the pressure applied is less than or equal to or anynumber in between 15 psi, 16 psi, 17 psi, 18 psi, 19 psi, 20 psi, 21psi, 22 psi, 23 psi, 24 psi, 25 psi, 26 psi, 27 psi, 28 psi, 29 psi, 30psi, 31 psi, 32 psi, 33 psi, 34 psi, 35 psi, 36 psi, 37 psi, 38 psi, 39psi, 40 psi, 41 psi, 42 psi, 43 psi, 44 psi, 45 psi, 46 psi, 47 psi, 48psi, 49 psi, 50 psi, 51 psi, 52 psi, 53 psi, 54 psi, 55 psi, 56 psi, 57psi, 58 psi, 59 psi, or 60 psi.

In some embodiments the plunger force is less than or equal to or anynumber in between 4 lbf, 5 lbf, 6 lbf, 7 lbf, 8 lbf, 9 lbf, 10 lbf, 11lbf, 12 lbf, 13 lbf, 14 lbf, 15 lbf, 16 lbf, 17 lbf, 18 lbf, 19 lbf, 20lbf, 21 lbf, 22 lbf, 23 lbf, 24 lbf, or 25 lbf.

The infusion is done intravascularly and/or intramuscularly. Preferredinfusion is intravascular, more preferably intra-arterial mostpreferably intra-coronary using a catheter coated with a biocompatiblematerial. The intracoronary delivery may be in a single dose or multipledoses or a single dose on different days or a multiple dose on differentdays. Doses are beneficially administered for not more than two minutesto prevent further ischemic damage, and may each be followed by aresting period during which the infusion site is exposed to blood flow,such as a resting period of 1, 2, 3, 4, or 5 minutes, and doses may bedelivered singly, or two at a time, three at a time, four at a time, ormore than four at a time, consecutively at an administration period,which may be followed by 3 mL of chaser solution, in some embodimentsnot exceeding 2.5 mL/min flow rate, to evacuate any remaining cells fromthe catheter lumen, and which may constitute a single administrationperiod or may be repeated at multiple administration periods such asadministration periods on successive days, or separated by 1, 2, 3, 4,5, 6, 7, or more than 7 days. That is, one may administer one dose fornot more than 2 minutes followed by a resting period of 1 minute, or onemay administer one dose for not more than 2 minutes followed by aresting period of 2 minutes, or one may administer one dose for not morethan 2 minutes followed by a resting period of 3 minutes, or one mayadminister one dose for not more than 2 minutes followed by a restingperiod of 4 minutes, or one may administer one dose for not more than 2minutes followed by a resting period of 5 minutes, or one may administertwo doses for not more than 2 minutes each followed by a resting periodof 1 minute, or one may administer two doses for not more than 2 minuteseach followed by a resting period of 2 minutes, or one may administertwo doses for not more than 2 minutes each followed by a resting periodof 3 minutes, or one may administer two doses for not more than 2minutes each followed by a resting period of 4 minutes, or one mayadminister two doses for not more than 2 minutes each followed by aresting period of 5 minutes, or one may administer three doses for notmore than 2 minutes each followed by a resting period of 1 minute, orone may administer three doses for not more than 2 minutes each followedby a resting period of 2 minutes, or one may administer three doses fornot more than 2 minutes each followed by a resting period of 3 minutes,or one may administer three doses for not more than 2 minutes eachfollowed by a resting period of 4 minutes, or one may administer threedoses for not more than 2 minutes each followed by a resting period of 5minutes, or one may administer four doses for not more than 2 minuteseach followed by a resting period of 1 minute, or one may administerfour doses for not more than 2 minutes each followed by a resting periodof 2 minutes, or one may administer four doses for not more than 2minutes each followed by a resting period of 3 minutes, or one mayadminister four doses for not more than 2 minutes each followed by aresting period of 4 minutes, or one may administer four doses for notmore than 2 minutes each followed by a resting period of 5 minutes, andthe total administration may be followed by 1 mL, 2 mL, 3 mL or morethan 3 mL of chaser solution to evacuate any remaining cells from thecatheter lumen in a single administration period.

For illustration, 4 doses of 5 mL each (total 20 ml) cellular productmay infused at 2.5 mL per minute, followed by a resting period of 1-5minutes, over a period of 12 to 28 min combined, followed by 3 mL ofchaser solution infused at 2.5 mL per minute to evacuate any remainingcells from the catheter lumen in a single administration period.

In a first set of human clinical trials, it was found that asignificantly improved response after cardiac ischemia could be obtainedusing the formulations of cell populations described herein. Improvementafter cardiac ischemia is clinically measured by observing animprovement in LVEF. A cell population comprising bone-marrow-derivedstem cells was prepared as described herein having a viscosity of lessthan or equal to or anything in between 1.0 cP through 5.0 cP (e.g., 1.0cP, 1.5 cP, 2.0 cP, 2.5 cP, 3.0 cP, 3.5 cP, 4.0 cP, 4.5 cP, or 5.0 cP oranywhere in between). The cell population was produced through a methodof making a regenerative cell composition comprising: (a) providing afirst cell population that comprises bone marrow stem cells; (b) mixingan anticoagulant with said first cell population so as to produce acomposition comprising said first cell population and anticoagulant; (c)enriching bone marrow stem cells within said composition comprising saidcell population and anticoagulant so as to produce a second cellpopulation that comprises enriched bone marrow stem cells; (d) isolatinga fraction comprising said second cell population that comprisesenriched bone marrow stem cells; and (e) adjusting the viscosity of saidfraction using a liquid to a viscosity of not more than 5.0 cP measuredat 37° C. so as to make said regenerative cell composition. Thisformulation was provided to patients suffering from cardiac ischemia bycatheter at a flow-rate of less than 2.5 mL/min (e.g., a number lessthan or equal to or in between 0.5 mL/min, 1.0 mL/min, 1.5 mL/min, 2.0mL/min, or 2.5 mL/min). The LVEF of the patient was determined over timeand it was found that the provision of the cell population comprisingbone marrow-derived stem cells prepared as described herein provided a4-fold improvement of LVEF as compared to conventional bonemarrow-derived stem cell populations (see, for a survey of conventionalstem cell composition performance, Delewi et al., (2013), “Impact ofintracoronary bone marrow cell therapy on left ventricular function inthe setting of ST-segment elevation myocardial infarction: acollaborative meta-analysis” European Heart Journal, 2013 Sep. 11,doi:10.1093/eurheartj/eht372).

In a second set of human clinical trials, it was found that asignificantly improved response after critical limb ischemia could beobtained using the formulations of cell populations described herein.Improvements of critical limb ischemia is clinically measured byobserving an improvement in amputation-free survival rate. A cellpopulation comprising bone marrow-derived stem cells was prepared asdescribed herein having a viscosity of less than or equal to or anythingin between 1.0 cP through 5.0 cP (e.g., 1.0 cP, 1.5 cP, 2.0 cP, 2.5 cP,3.0 cP, 3.5 cP, 4.0 cP, 4.5 cP, or 5.0 cP or anywhere in between). Thecell population was produced through a method of making a regenerativecell composition comprising: (a) providing a first cell population thatcomprises bone marrow stem cells; (b) mixing an anticoagulant with saidfirst cell population so as to produce a composition comprising saidfirst cell population and anticoagulant; (c) enriching bone marrow stemcells within said composition comprising said cell population andanticoagulant so as to produce a second cell population that comprisesenriched bone marrow stem cells; (d) isolating a fraction comprisingsaid second cell population that comprises enriched bone marrow stemcells; and (e) adjusting the viscosity of said fraction using a liquidto a viscosity of not more than 5.0 cP measured at 37° C. so as to makesaid regenerative cell composition. This composition was provided topatients suffering from critical limb ischemia by cannula at a flow-rateof less than 2.5 mL/min (e.g., a number less than or equal to or inbetween 0.5 mL/min, 1.5 mL/min, 2.0 mL/min, or 2.5 mL/min). Theamputation-free survival rate of the patients was determined over timeand it was found that the provision of the cell population comprisingthe bone marrow-derived stem cells prepared as described herein provideda 25% improvement of amputation-free survival as compared toconventional bone marrow-derived stem cell populations as compared tostudies including the Harvest Technologies CLI study, TherapeuticAngiogenesis using Cell Transplantation in critical limb ischemia (TACTstudy), and an Amann et al Study as listed at Example 6, below.

Thus, through the practice of the disclosure herein, one may observedramatic improvements over results observed previously with conventionalbone marrow-derived stem cell populations for the treatment of ischemia(e.g., cardiac ischemia and limb ischemia). One can observe in someinstances a percent improvement in left ventricle ejection fraction(LVEF) of 2×, 3×, 4×, or 5×, or more than 5× as compared to aconventional preparation of a cell population comprising bone marrowstem cells. Similarly, one can observe in some instances a percentimprovement in amputation free survival rate among limb ischemiapatients of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or greater than 24% ascompared to a conventional preparation and delivery of a cell populationcomprising bone marrow stem cells.

EXAMPLES Example 1 Bone Marrow Aspiration

The bone marrow used as a source material was autologous. The bonemarrow from which stem cells are isolated was selected from the groupconsisting of iliac crest, femora, tibiae, spine, and ribs, or othermedullary spaces in bone. The preferred bone marrow source was the iliaccrest. Before starting the aspiration of bone marrow, the anticoagulantsolution was filled into aspiration syringes. Each vial contains 250 mgof bivalirudin. 5 ml of sterile water was added for injection and thevial was gently swirled until all material was dissolved. The solutionwas poured into a 20 ml sterile syringe. The syringe was filled with0.9% sodium chloride for injection to the 12.5 ml mark, to yield a finalconcentration of 20 mg/ml. Next, three sterile 20 ml syringes werefilled with 2 ml of 20 mg/ml bivalirudin solution. 18 ml of bone marrowwere aspirated in each syringe to make a final volume of 20 ml. Thefinal concentration of bivalirudin, along with aspirated bone marrow, ineach syringe was 2 mg/ml. The aspiration needle/trocar used was 11gauge. Bone marrow was aspirated in the syringe or syringes pre-filledwith anticoagulant solution for a final cumulative volume between 120 mlto 180 ml. The aspiration syringe used was a 20 ml syringe.

Modifications to the above method may be made without departing from theinvention. For example, entry may be made into more than one-site toaspirate bone marrow. Some amount of anti-coagulant solution may beinjected into bone marrow before aspirating it. In the next step, theaspirated bone marrow is thoroughly mixed with anticoagulant solution byrolling gently between the palms of hands. The bone marrow is thentransferred into a device for separating the components of bone marrow,the art of which is known.

Example 2 Bone Marrow Cell Analysis and Processing

The aspirated bone marrow was analyzed (both at pre- and post-processingstep) and processed, in a point-of-care environment. Initially 120 ml ofanti-coagulated bone-marrow was harvested. Nucleated cells present perml of sample were rapidly analyzed using a small sample (less than 0.5ml) of it. If needed, 30-60 ml of additional bone marrow may beharvested for processing in order to get the desired infusion cell dose.The processing technology comprises the following components; (i) asterile, disposable bone marrow processing device that can processvariable volumes of bone-marrow up to 400 mL, (ii) a control modulehaving at least one gravitational and one or more Infra-red (IR)/opticalsensors coupled with a microprocessor and motors that control the flow,movement, and compartmentalization of the separated fractions into adisposable device or compartment, (iii) a docking station for thecontrol module that transfers processing data from the control modulefirmware to a computer and (iv) a centrifuge with programmableparameters.

Example 3 Bone Marrow Viscosity and Analysis

The concentrated stem cells infusate viscosity was titrated to ensureshear stresses are controlled during trans-catheter delivery. Aviscometer, possibly including but not required ancillary software,determined the stem cell concentrate viscosity in units (for examplecentipoise), and calculated the required plasma volume required to yielda final infusate in the preferred range of 1.8-5 cP at 98.6° Fahrenheitor 37° Celsius. A preferred infusate viscosity was 3.1 cP-5.0 cP, andthe most preferred infusate viscosity was 1.8 cP-3.0 cP, or 2.0 cP-2.5cP. The automated or manual processing technology having produced thestem cell fraction, plasma fraction, and RBC fraction in less than 30minutes at the patient's bedside served as the source of the autologousplasma.

Example 4 Protocol for Infusion of Cellular Product

The aspirated and processed bone marrow was infused as an adjuvant toprimary care. The bone-marrow stem cell, with or without factors,concentrate was infused after ischemic conditioning of the tissue. Theblood flow to the affected area was stopped using an over the wireinflatable balloon inflated to reduce blood flow distally to the balloon80-99.99% for a period of no more than three minutes, preferably oneminute and most preferred for two minutes. Following a short period oftissue ischemia, cells were infused at a controlled rate, no more than2.5 ml per minute. The infusion was done intravascularly and/orintramuscularly. Preferred infusion is intravascular, more preferablyintra-arterial most preferably intra-coronary using a catheter coatedwith a biocompatible material. The intracoronary delivery was in asingle dose or multiple doses or a single dose on different days or amultiple dose on different days. For illustrations, 4 doses of 5 ml each(total 20 ml) cellular product was infused over a period of 12 to 28min, followed by 3 ml of chaser solution to evacuate any remaining cellsfrom the catheter lumen.

Example 5 Safe Study of Autologous Bone Marrow Concentrate Enriched inProgenitor Cells (BMCEPC) as an Adjuvant, in the Treatment of AcuteMyocardial Infarction

A 43 year old male, non-diabetic, normo-tensive, nonobese, smokerpresented with a history of two hours of chest pain and symptomatic ofan AMI into the emergency department. On admission, a 2 mm ST segmentelevation in the anterior leads was observed, and AMI was furtherconfirmed with biochemical blood tests. The patient's Left VentricularEjection Fraction, LVEF, was estimated to be around 35% by bedside 2DECHO. Primary percutaneous coronary intervention (PCI) was performedusing a routine technique, and a single drug-eluting stent was deployedin the proximal LAD with TIMI-3 grade flow results. Post-PCI, thepatient's LVEF remained <40% at the 120 hour time-point as measured bymultigated acquisition (MuGA) and ECHO, which met our inclusion criteriaand is predictive of a higher than acceptable one year mortality rate.LVEF is one of the key indications of mortality rates post MI with areduced LVEF being a risk factor for both sudden and non-sudden death,with the odds ratio for 1-year mortality after MI at 9.48 for patientswith LVEF ≦30% compared with patients with LVEF >50%, 2.94 for patientswith LVEF 30-40%, whereas the risk was not significantly increased inpatients with LVEF 40-50%.

The patient was advised that he met the inclusion criteria for theAMIRST clinical trial program using his own (autologous) bone marrowstem cells. The clinical trial was registered with clinicaltrials.gov(NCT01536106) and was approved by the Institutional Ethics Committee(IEC) (IEC Approval # TIEC/2011/32/02) and Institutional Committee forStem Cell Research (IC-SCRT). The Patient, Primary Investigator andClinical Investigator concurred, and consent was obtained. On the sixthday post PTCA/stent implant, the patient was transferred to the heartcatheterization laboratory, and the AMIRST (Acute Myocardial InfarctionRapid Stem cell Therapy) protocol was completed as disclosed in thesummary and detailed description herein.

The entire procedure was completed within 90 minutes, taking 30 minuteslonger than anticipated as the vascular surgeon trained for collectingthe bone marrow was delayed. As a preliminary safety study prior to fullsubject enrollment, the patient was followed up for 24 months, andevaluated with standard diagnostic metrics. No major adverse cardiacevents (MACE) including re-hospitalization were reported, demonstratingthe safety of this adjuvant treatment. The patient's LVEF improved from36% (Day 0) at the time of the AMIRST treatment to 60.3% at 24 monthspost-AMIRST intervention. It is noted that in this study MuGA was usedas the enrollment measurement technique. Caution should be taken incomparing MuGA and MRI LVEF results; however the enrollment LVEF wasconfirmed via a secondary method i.e. ECHO and the 3-month and 24-monthsLVEF results were confirmed by the same radiology team.

Upon written consent, and within a maximum window of 10 days post MI,the patient was taken to the heart catheterization laboratory (operatingroom suite) on Day 6 post-PCI, mildly to moderately sedated using 0.2mcg/kg of Fentanyl, and 120 mL of bone marrow was aspirated from thepatient's iliac crest using an 11-gauge Jamshidi needle optimized forcell harvest. Careful bone marrow aspiration technique was employed toreduce peripheral blood contamination in the aspirate. Following theaspiration, the bone marrow was processed employing our proprietarypoint-of-care technology platform to produce bone marrow concentrateenriched in progenitor cells (BMC_(E)PC). The cellular product containeda total of 3.54×10⁸ BMMNCs. A guide-wire was introduced into the femoralartery followed by a double lumen ultra-low profile PTA intracoronarycatheter, and the patient had four separate induced ischemia/progenitorcell infusions using the “stop-flow” technique before the entire optimaldose of nucleated cells was distally delivered to the stent in the LAD.The complete process was accomplished in 90 minutes at patient'sbedside. The patient's hematological and biochemical parameters arelisted in Table 4.

TABLE 4 Clinical Laboratory Values Pre and Post BMCePC Therapy PreBMCePC Post BMCePC Infusion Infusion Day −2 Day 0 Hemaglobin (g/dL) 13.513.8 RBC Count (×10(6)/uL) 4.84 4.92 Platelet Count (×10(3)/mL) 110 120Creatinine, Serum (mg/dL) 0.8 0.9 Urea Nitrogen (mg/dL) 13 12 CreatineKinase, Serum 106 188 N-Terminal pro-B Type 731.3 — Natriuretic Peptide(pg/mL)

There were no adverse events (AE) or serious adverse events (SAE)reported during the procedure. The patient remained hospitalized fortelemetry an additional 24 hours post cell transplant, and released withstandard cardiac therapeutics as listed in Table 5.

TABLE 5 Medications Prescribed on Discharge post BMCePC Therapy DrugDose Frequency Drug Class Ecosprin 150 mg Oral, Once Daily Anti-plateletDeplatt  75 mg Oral, Twice Daily Anti-platelet Rosuvas  20 mg Oral, OnceDaily Statin Pantacid  40 mg Oral, Once Daily Dyspepsia

The patient was scheduled for follow-up on 1, 2, 3, 6, 12 and 24 monthsto assess the primary endpoints of safety and the secondary endpoints ofefficacy that includes MACE, LVEF, cardiac output, cardiac remodelingand quality of life assessments. The 12-month follow-up could not becompleted due to non-availability of patient, but all other follow-uppoints were completed. No Major Adverse Cardiac Events (MACE) orre-hospitalization events were reported. The patient continued to followa normal life, after 2 weeks post the AMIRST procedure. At the 1-monthfollow-up, HOLTER monitoring was performed for 23 hours and 6 minutes.No ventricular ectopics were observed, and the heart variability wasnormal. The slowest episode of bradycardia (HR 53 bpm, 1 min 13 sec) wasobserved at midnight and the fastest episode of tachycardia (HR 141 bpm,1 min 2 sec) was observed in the afternoon. Cardiac imaging wasperformed at each follow-up. The cardiac chambers appeared normal withno signs of pericardial effusion.

Overall, the study demonstrated safety of the bone marrow aspiration,processing and infusion methodology as disclosed herein in an acute lowLVEF infarct patient post PTCA. FIGS. 2A-D show cardiac MR imagesobtained 3- and 24-months post-BMC_(E)PC infusion. At the 24-monthfollow-up, the cardiac MR findings were summarized as “Basal andmid-cavity anterior and anteroseptal and apical anterior and septal andapex myocardial post contrast sub-endocardial <25% to 50% with a focalspec of 75% transmural hyper-enhancement, consistent with ischemicinfarction.”

The pre-BMC_(E)PC infusion cardiac MR imaging was performed on adifferent instrument than the 3- and 24-months post-BMC_(E)PC infusion.Therefore, an absolute quantitative equivalency measurement of LVEFbetween the pre-treatment and 3-months post treatment should beevaluated cautiously. Also, the MuGA and ECHO scan results have a levelof user dependency, and each result should be cautiously interpreted.

Nevertheless, a considerable improvement in the LVEF has been noted overthe study period and between 3-months and 24-months follow-up. PostBMC_(E)PC infusion, the LVEF improved from 36% (Day 0) at the time ofthe AMIRST treatment to 55.3% at 3 months post-AMIRST intervention, andfurther improved from 55.3% to 60.3% at 24 months post-AMIRSTintervention, as shown in Table 6. That is, the patient showed a 67.5%increase from day 0 through 24 months. This degree of improvement isconsidered atypical for a patient having suffered an ST elevatedmyocardial infarction with an ejection fraction below 40% postreperfusion (stenting).

TABLE 6 Time Endpoints: Left Ventricular Ejection Fraction (LVEF) Valuesand Safety Measurement Time Method Value MACE Time 0 Days 2D Echo    36%N/A Time 6 Days MuGA  <40% N/A Time 7 Days IC Angiogram  <40% None Time3 Months F/U cMRI  55.30% None Time 24 Months F/U cMRI  60.30% None

The cardiac output (volumetrics) also showed an improvement from 2.7l/min to 3.4 l/min over the same period, and is a secondary endpoint. Noreduction in scar size was observed with a heart mass of 115.5 gms and ascar mass of 11.5 gms (approximately 11%).

As previously disclosed (Delewi et al., (2013), “Impact of intracoronarybone marrow cell therapy on left ventricular function in the setting ofST-segment elevation myocardial infarction: a collaborativemeta-analysis” European Heart Journal, 2013 Sep. 11,doi:10.1093/eurheartj/eht372), a meta-analysis of 16 studies including1641 patients (984 cell therapy, 657 controls) reported that absoluteimprovement in LVEF was increased 2.55% among BMC-treated patientscompared with controls: [95% confidence interval (CI) 1.83-3.26, p valueof 0.001]. Cell therapy significantly reduced LVEDVI and LVESVI (23.17mL/m2, 95% CI: 24.86 to 21.47, p=0.001; 22.60 mL/m2, 95% CI 23.84 to21.35, p value of 0.001, respectively). Among patients under age 55, theabsolute improvement in LVEF was increased to a slightly greater 3.38%,and among patients with baseline LVEF <40%, the absolute improvement inLVEF was increased again to a slightly greater 5.30%.

Accordingly, the 67.5% degree of improvement or 24.3 percentage pointsof improvement observed through practice of the disclosure herein ismore than 4-fold greater than the 5.30% level of improvement expectedfor the under 55 age cohort with baseline LVEF <40% to which the studypatient would be a member using conventional techniques that utilizecell populations having bone marrow stem cells.

Example 6 60 Minute Rapid Bedside Treatment Reduces Amputations inNo-Option Limb Ischemia Patients

Critical limb ischemia afflicts an estimated 2 million people combinedin the United States, European Union and Indian sub-continent, andresults in approximately 500,000 amputations each year. The overallprevalence (0.23%) and incidence (0.20%) in the United States increaseswith age and diabetes status, and 5 year mortality rate post limbamputation reaches nearly 50%.

A patient treatment protocol was completed as disclosed in the summaryand detailed description herein. Heparin was used as an anti-coagulant.Phase Ib clinical trial safety and efficacy results treating no-optionpatients suffering from critical limb ischemia CLIRST (Critical LimbIschemia Rapid Stem cell Therapy) treatment are presented. The trialachieved both its primary safety endpoint and secondary efficacyendpoints at 12 months having no serious adverse events determined to berelated to the therapy, and achieving statistical significance inamputation free survival rates (82.4%), pain reduction (mean VAS scorepre-therapy 7.8±0.97 and 12 month follow-up 0.2±0.58 on a scale of 0-10,p=0.0005), 6-minute walking distance (mean distance pre-therapy of 14.5meters±37.57 and 12 month follow-up of 157 meters±100.92, p=0.0039),open wound healing (11 patients had gangrene with or without ulcerationpre-treatment and all patients had neither gangrene nor ulceration at 12month follow-up), and TcPO2 (transcutaneous oxygen pressure) levels(mean pre-therapy of 14.66±6.93 improved to 35.75±17.04, p=0.0032.

Additionally, and not demonstrated in earlier studies, vasculogenesisimprovements in the treated leg were seen. Improvement was observed inboth collateral vessel number (p=0.0156 in distal thigh, p=0.0313 inproximal leg), and vessel size (distal thigh, p=0.0156 and proximal leg,p=0.0625) were observed. FIGS. 3A and 3B depict images of improvement incollateral vessel size and number in an patent treated in this study.

Quantification of these results for a single exemplary individual isgiven in Table 7, presenting Qualitative and Quantitative collateralvessel data for subject EHIRC/CLI-STEM-002 as shown in pre and postangiograms, FIGS. 3A and 3B. An overview of aggregate results is givenin Table 8, below, presenting data related to the number of collateralvessels and the collateral vessel size before and 12 months afteradministration of a composition herein through the practice of methodsherein.

Categories used for the quantification were scored as follows: CategoryScoring (No. of collateral Vessels): 0: No collateral vessels; 1: 1-3Collateral vessels; 2: 4-7 Collateral Vessels; 3: =>8 CollateralVessels; Grade Scoring (Size of Collateral Vessel): 0: No collateralvessels; 1: =<5 Small; 2: >5 Small; 3: =<5 large±Small; 4: >5large±Small.

TABLE 7 Category Grade (No. of Collaterals) (Size of Collateral Vessels)Pre- Post- Pre- Post- Vessel Intervention Intervention p ValueIntervention Intervention p Value Proximal thigh 0 0 0.1250 0 0 0.2500Mid thigh 1 3 0.0547 1 3 0.1563 Distal thigh 1 3 0.0156 1 3 0.0156Proximal leg 0 2 0.0313 0 3 0.0625 Mid leg 0 1 0.5000 0 1 0.7500 Distalleg 0 0 >0.9999 0 0 >0.9999

TABLE 8 Summary table of CT Angiography vessel collateral grading of 14critical limb ischemia no-option patients (pre-intervention and 12 monthpost intervention) with the BMC_(E)PC infusion intramuscularly. Numberof Collateral Vessel Size of Collateral Vessel Category Grade Pre Post pPre Post Parameters Mean (SD) Mean (SD) Value Mean (SD) Mean (SD) pValue Thigh Proximal Thigh 0.42 (0.793) 0.75 (1.055) 0.1250 0.58 (1.165)1.00 (1.348) 0.2500 Mid Thigh 0.58 (0.793) 1.25 (1.357) 0.0547 0.58(0.793) 1.17 (1.193) 0.1563 Distal Thigh 0.42 (0.669) 1.42 (1.311)0.0156 0.33 (0.492) 1.33 (1.155) 0.0156 Leg Proximal Leg 0.25 (0.622)1.08 (1.084) 0.0313 0.17 (0.389) 1.08 (1.165) 0.0625 Mid Leg 0.33(0.778) 0.67 (1.073) 0.5000 0.25 (0.622) 0.42 (0.669) 0.7500 Distal Leg0.42 (0.996) 0.42 (0.996) >0.9999 0.25 (0.622) 0.25 (0.622) >0.9999

The open label single study enrolled 17 patients, of which 14successfully achieved the 12 month major amputation free endpointtarget. Patients were treated with a mean cell dose of BMC_(E)PC (bonemarrow concentrate enriched progenitor cells) of 8.04×10⁸ (±3.65×10⁸)cells in a 20 ml final product, which was injected intramuscularly inthe lower afflicted leg. Additionally, patient participants in the studydemonstrated an improved number of vessels in the mid-thigh (p=0.0547),distal thigh (p=0.0156); and proximal leg (p=0.0313), and improvedvessel size in the distal thigh (p=0.0156), and proximal leg (p=0.0625).

A summary of amputation free survival rate 12 months post treatment withthe BMC_(E)PC infusion intramuscularly is given in Table 9, below.

TABLE 9 n (%) [N = 17] Total Major Limb Amputation free survival rate 14(82.35) Total Amputations  5 (29.41) Major Amputation  3 (17.6) MinorAmputation  2 (11.8)

A comparison of amputation-free survival rates of the present exampleand treatments known in the art from other studies is given in Table 10.

TABLE 10 BONE BED- AMPUTATION CLAUDICATION MARROW SIDE FREE PAIN SCORESTUDY CELLS PROCESSING CELL DOSE SURVIVAL (VAS) CLIRST BMC - Yes   1 ×10⁸ Major AFS = Before: 7.8; 12 (Toti, Bedside 82.35% Mo After: 0.5Example Integrated Kit 6 herein) n = 17 Harvest BMC - Yes 3.3 × 10⁹Major AFS = n/a Tech. Bedside 58% n = 51 TACT BMC - Ficoll No (lab - 3hrs) 1.6 × 10⁹ 3 Yr AFS 60% Before = 6 Study Post = 2 p = .0078 AmannBMC - Ficoll No 1.1 × 10⁹ 1 Yr AFS 53% Not measured. Study n = 12 Usedanalgesic (2 arm BMC - Yes 3.0 × 10⁹ 1 Yr AFS 58% consumption as anstudy) Bedside endpoint BONMET-1 n = 39

As observed in a comparison of the results of previous studies aspresented in Table 7, the amputation free survival rate of 82% observedthrough the practice of the disclosure herein is almost 25% greater thanthe rate reported through previous studies using techniques that utilizeconventional cell populations having bone marrow stem cells.

While the foregoing written description of the invention enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific embodiment, method, and examples herein. The inventionshould therefore not be limited by the above described embodiment,method, and examples, but by all embodiments and methods within thescope and spirit of the invention as claimed.

What is claimed is:
 1. A point-of-care method of ameliorating ischemiaor a condition associated with ischemia in a subject comprising:identifying a subject having an ischemia or a condition associated withischemia; providing to said subject a composition comprising: (a) afirst cell population that comprises CD34⁺ and CD34⁻ bone marrow stemcells, (b) an anticoagulant, and (c) autologous plasma, wherein thecomposition has a viscosity of 1.5 to 5.0 centipoise (cP) measured at37° C., and wherein said composition is provided to said subject at arate of 1.0 mL per minute.
 2. The method of claim 1, wherein thecomposition is provided to said subject not more than 2 hours after saidfirst cell population is obtained.
 3. The method of claim 1, whereinsaid composition is provided to said subject not more than 90 minutesafter said first cell population is obtained.
 4. The method of claim 1,wherein said composition is provided to said subject not more than 60minutes after said first cell population is obtained.
 5. The method ofclaim 1, wherein the blood flow at an ischemic tissue of said subject isblocked by an inflatable balloon, wherein said balloon is inflated toblock at least 80% of blood flow distal to said balloon.
 6. The methodof claim 1, wherein said composition provided to said subject is notsubjected to a maximum shear of more than 9101/second.
 7. The method ofclaim 1, wherein said composition is provided to said subject through acatheter having a lumen size of approximately 0.36 mm.
 8. The method ofclaim 1, wherein said composition is provided intravascularly,intra-arterially or intra-coronarily.
 9. The method of claim 1, whereinthe ischemia is cardiac ischemia.
 10. The method of claim 9, furthercomprising measuring in said subject an improvement in left ventricleejection fraction, an improvement in cardiac output, a reduction incardiac scar formation, or an improvement in cardiac remodeling.
 11. Themethod of claim 1, further comprising measuring in said subject anincreased blood flow, an improvement in left ventricle ejectionfraction, an improvement in cardiac output, a reduction in cardiac scarformation, an improvement in cardiac remodeling, an increase inangiogenesis, an increase in vascularity, an improvement in neural scarformation, or an increase in amputation-free survival rate.
 12. Themethod of claim 1, wherein said composition has a viscosity of 1.8 to5.0 centipoise (cP) measured at 37° C.
 13. The method of claim 1,wherein said CD34⁻ bone marrow stem cells comprise mesenchymal stemcells or stromal cells.
 14. The method of claim 13, wherein saidmesenchymal stem cells or stromal cells are lineage negative/dim, CD45negative/dim and CD73 positive cells.
 15. The method of claim 1, whereinsaid first cell population comprises hematopoietic stem cells,mesenchymal stem cells, strotnal cells, endothelial progenitor cells,CXCR4 positive cells, or a combination thereof.
 16. The method of claim1, wherein said composition comprises red blood cells.
 17. Apoint-of-care method of ameliorating ischemia or a condition associatedwith ischemia in a subject comprising: identifying a subject having anischemia or a condition associated with ischemia; introducing bonemarrow aspirate from said subject into a bone marrow processing system;isolating a stem cell fraction, a plasma fraction, and a red blood cellfraction from said disposable bone marrow processing device; addingplasma from said plasma fraction and red blood cells from said red bloodcell fraction to said stem cell fraction to obtain a composition with aviscosity of 1.5 to 5.0 centipoise (cP) measured at 37° C.; andproviding said composition with a viscosity of 1.5 to 5.0 centipoise(cP) measured at 37° C. to said subject.
 18. The method of claim 17,wherein said composition comprises an anticoagulant.
 19. The method ofclaim 17, wherein said stem cell fraction comprises CD34⁺ and CD34⁻ bonemarrow stem cells.
 20. The method of claim 19, wherein said CD34⁻ bonemarrow stem cells comprise mesenchymal stem cells or stromal cells. 21.The method of claim 20, wherein said mesenchymal stem cells or stromalcells are lineage negative/dim, CD45 negative/dim and CD73 positivecells.
 22. The method of claim 17, wherein said composition has aviscosity of 1.8 to 5.0 centipoise (cP) measured at 37° C.
 23. Themethod of claim 17, wherein said composition is provided to said subjectat a rate of 1.0 to 2.0 mL per minute.
 24. The method of claim 17,wherein said composition is provided to said subject at a rate of 1.0 mLper minute.
 25. The method of claim 17, wherein said composition isprovided to said subject not more than 2 hours after said first cellpopulation is obtained.
 26. The method of claim 17, wherein saidcomposition is provided to said subject not more than 90 minutes aftersaid first cell population is obtained.
 27. The method of claim 17,wherein said composition is provided to said subject not more than 60minutes after said first cell population is obtained.
 28. The method ofclaim 17, wherein the blood flow at an ischemic tissue of said subjectis blocked by an inflatable balloon, wherein said balloon is inflated toblock at least 80% of blood flow distal to said balloon.
 29. The methodof claim 17, wherein said composition provided to said subject is notsubjected to a maximum shear of more than 9101/second.
 30. The method ofclaim 17, wherein said composition is provided to said subject through acatheter having a lumen size of approximately 0.36 mm.
 31. The method ofclaim 17, wherein said composition is provided intravascularly,intra-arterially or intra-coronarily.
 32. The method of claim 17,further comprising measuring in said subject an increased blood flow, animprovement in left ventricle ejection fraction, an improvement incardiac output, a reduction in cardiac scar formation, an improvement incardiac remodeling, an increase in angiogenesis, an increase invascularity, an improvement in neural scar formation, or an increase inamputation-free survival rate.
 33. The method of claim 17, wherein theischemia is cardiac ischemia.
 34. The method of claim 33, furthercomprising measuring in said subject an improvement in left ventricleejection fraction, an improvement in cardiac output, a reduction incardiac scar formation, or an improvement in cardiac remodeling.
 35. Apoint-of-care method of ameliorating ischemia or a condition associatedwith ischemia in a subject comprising: identifying a subject having anischemia or a condition associated with ischemia; introducing bonemarrow aspirate from said subject into a bone marrow processing systemthat comprises: (a) a sterile, disposable bone marrow processing device,(b) a control module comprising a gravitational sensor and an infra-redoptical sensor coupled with a microprocessor and motor, which areconfigured to control the flow, movement, and compartmentalization ofseparated fractions of bone marrow aspirate, (c) a docking station thattransfers processing data from the control module to a computer, and (d)a centrifuge; isolating a stem cell fraction, a plasma fraction, and ared blood cell fraction from said disposable bone marrow processingdevice; adding plasma from said plasma fraction and red blood cells fromsaid red blood cell fraction to said stem cell fraction to obtain acomposition with a viscosity of 1.5 to 5.0 centipoise (cP) measured at37° C.; and providing said composition with a viscosity of 1.5 to 5.0centipoise (cP) measured at 37° C. to said subject.
 36. The method ofclaim 35, wherein said composition comprises an anticoagulant.
 37. Themethod of claim 35, wherein said stem cell fraction comprises CD34⁺ bonemarrow stem cells and mesenchymal stem cells.
 38. The method of claim35, wherein said stem cell fraction comprises stromal cells.
 39. Themethod of claim 38, wherein said stromal cells are lineage negative/dim,CD45 negative/dim and CD73 positive cells.
 40. The method of claim 35,wherein said composition has a viscosity of 1.8 to 5.0 centipoise (cP)measured at 37° C.
 41. The method of claim 35, wherein said compositionis provided to said subject at a rate of 1.0 to 2.0 mL per minute. 42.The method of claim 35, wherein said composition is provided to saidsubject at a rate of 1.0 mL per minute.
 43. The method of claim 35,wherein said composition is provided to said subject not more than 2hours after said first cell population is obtained.
 44. The method ofclaim 35, wherein said composition is provided to said subject not morethan 90 minutes after said first cell population is obtained.
 45. Themethod of claim 35, wherein said composition is provided to said subjectnot more than 60 minutes after said first cell population is obtained.46. The method of claim 35, wherein the blood flow at an ischemic tissueof said subject is blocked by an inflatable balloon, wherein saidballoon is inflated to block at least 80% of blood flow distal to saidballoon.
 47. The method of claim 35, wherein said composition providedto said subject is not subjected to a maximum shear of more than9101/second.
 48. The method of claim 35, wherein said composition isprovided to said subject through a catheter having a lumen size ofapproximately 0.36 mm.
 49. The method of claim 35, wherein saidcomposition is provided intravascularly, intra-arterially orintra-coronarily.
 50. The method of claim 35, further comprisingmeasuring in said subject an increased blood flow, an improvement inleft ventricle ejection fraction, an improvement in cardiac output, areduction in cardiac scar formation, an improvement in cardiacremodeling, an increase in angiogenesis, an increase in vascularity, animprovement in neural scar formation, or an increase in amputation-freesurvival rate.
 51. The method of claim 35, wherein the ischemia iscardiac ischemia.
 52. The method of claim 51, further comprisingmeasuring in said subject an improvement in left ventricle ejectionfraction, an improvement in cardiac output, a reduction in cardiac scarformation, or an improvement in cardiac remodeling.